This is a resubmitted application. We have revised and strengthened the proposal in light of significant new results and in response to the reviewers'critiques. Arenaviruses are endemic in rodent populations worldwide and can be transmitted to humans to cause acute hemorrhagic fevers. Prophylactic vaccines and effective therapies are lacking and urgently needed to address public health and biodefense concerns. Intervention strategies that target the viral envelope glycoprotein (GP-C) and virus entry into target cells are thus attractive. The arenavirus GP-C is unusual in that the mature complex retains its cleaved signal peptide (SSP) as an essential subunit in association with the conventional receptor-binding (G1) and transmembrane fusion (G2) proteins. Our preliminary results have shown that SSP interacts with the ectodomain of G2 to modulate pH-dependent membrane-fusion activity of the GP-C complex, and to form the target for a class of newly discovered small-molecule fusion inhibitors. SSP also associates with the cytoplasmic domain of G2 to regulate intracellular transport of the GP-C complex to the cell surface for virion assembly and budding. Our recent evidence indicates that the cytoplasmic interaction between SSP and G2 is mediated through a novel intersubunit zinc-finger motif. The broad, long-term goal of this project is to define the structure-function relationships in the tripartite GP-C complex that promote virion assembly and virus entry, in order to exploit the unique features of GP-C in the design of effective antiviral therapeutics. Towards this goal, we propose three specific aims: i) To characterize in molecular detail the interaction between SSP and the ectodomain of G2, and to understand the mechanism of action of the newly discovered small-molecule arenavirus fusion inhibitors;ii) To identify the molecular requirements for zinc binding in an unusual zinc-finger motif bridging SSP and the cytoplasmic domain of G2, and to characterize its role in the GP-C complex;and iii) To investigate the role of SSP and the intersubunit zinc finger in virion morphogenesis. PUBLIC HEALTH RELEVANCE: Arenaviruses are responsible for hemorrhagic fevers with high mortality. Effective therapies against Lassa fever, Argentine hemorrhagic fever and others are urgently needed to address ongoing public health and Category A biodefense concerns. The goal of this project is to characterize the unusual arenavirus envelope glycoprotein in molecular detail to guide strategies towards the development of prophylactic and therapeutic agents.